Plasma high density lipoprotein levels are inversely related to atherosclerosis and its complications. The ATP binding cassette transporter ABCA1 mediates cholesterol efflux from macrophage foam cells to apoA-1. ABCA1 trafficks from the cell surface to late endosomes and co-operates with the Niemann-Pick C1 (NPC1) molecule in promoting efflux of cholesterol from endosomes to apoA-1. ApoE-/-NPC1-/- mice display accelerated atherosclerosis, luminal thrombosis and medial erosion. NPC1-/- or modified LDL loaded macrophages have increased Cathepsin K expression and secretion, related to cholesterol accumulation in endosomes, and this is partly reversed by LXR activation, induction of ABCA1 and cholesterol efflux to apoA-1. Endosomal cholesterol accumulation seems to cause activation of a partial osteoclast differentiation program, similar to that induced by binding of RANK ligand to RANK (Receptor Activator of NFkB) on macrophages and requiring activation of p38 MAP kinase. In Aim1 we will further define the relationship between different endosomal cholesterol pools, induction of CatK and reversal by variants of ABCA1 with altered intracellular trafficking. We will evaluate the hypothesis that cholesterol accumulation in endosomes or membrane rafts leads to auto-activation of RANK and downstream signaling via p38 MAP kinase to induce CatK. In Aim 2 we will carry out a transgenic rescue of neurological disease in ApoE-/-NPC1-/- mice allowing longer term studies of atherosclerosis and its complications. We will introduce the CatK-/- trait into this background and overexpress the native CatK transgene in ApoE-/- mice in order to evaluate the hypothesis that CatK overexpression is necessary and sufficient to produce medial erosion and luminal thrombosis in ApoE-/- mice. We will also carry out a macrophage-specific KO of p38 MAP kinase in apoE-/- NPC1-/- mice to evaluate other potential downstream effects of augmented p38 MAP kinase signaling. Finally, we will determine if atherosclerosis and its complications are can be reversed in ApoE-/-NPC1-/- mice by adenovirus-apoA-1 expression and LXR activation. These studies should help to elucidate a mechanistic link between cholesterol accumulation in macrophages within specific cellular compartments and induction of CatK expression, leading to matrix degradation and breakdown of atherosclerotic plaques. These mechanistic insights may suggest new treatments for vulnerable human plaques such as LXR activators, CatK inhibitors or inhibitors of RANK signaling.